Bioconjugates of synthetic apelin polypeptides
a synthetic apelin and polypeptide technology, applied in the field of bioconjugates of synthetic apelin polypeptides, can solve the problems of acute decompensation of patients with chronic heart failure, cardiac contractility decline, and cardiac failure, and achieve the effect of increasing conformational constraints and increasing half-li
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example 1
Albumin-MPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R—P—R-L-C*—H—F-G-P-Nle-C*4-OH
[0538]To a solution of albumin (500 mg, Aldrich, lyophilized powder, from human serum) in 10 mL of PBS 1× buffer in a 15 mL tube was added a solution of TCEP hydrochloride (1.074 mg in bio-grade purified water) once. The resultant solution was shaked at it for 1 hr, then desalted and washed with two Amicon Ultra-4 centrifugal filters (30K MWCO). The filters were spinned at 4K g for 40 mins and the filtrates were discarded. 3 mL of bio-grade purified water was added to each filter for each wash (spinned at 14K g for 10 mins) and the wash process was repeated 3 times. The decapped HSA was dissolved in water (˜20 mL in total). The solution was transferred to a 50 mL Falcon tube, and lyophilized to give a crystalline powder (500 mg).
[0539]The pure product was analyzed by UPLC-MS (Analytical method F; measured: 66439.0; expected: 66437).
[0540]Determination of the Number of Fre...
example 2
Albumin-TPA-O2Oc-O2Oc-O2Oc-O2Oc-Q-R—P—R-L-C*—H—F-G-P-Nle-C*4-OH wherein TPA is: 3-mercaptopropanoic acid
[0554]A solution of decapped HSA (65.8 mg) in PBS buffer (1 mL) portionwise (100 ul in every 30 min) was added a solution of Peptide-Linker Construct 2 (8 mg) and sodium bicarbonate (8.92 uL, 1M) in PBS buffer (1 mL). After addition, the resultant solution was shaked at it overnight, then desalted and washed with 4 Amicon Ultra-0.5 centrifugal filters (10K MWCO). The filters were spinned at 13K g for 10 min and the filtrates were discarded. Bio-grade purified water was added to the top of each filter for each wash (spinned at 13K g for 10 mins) and the wash process was repeated 5 times. The conjugate was dissolved in water (4 mL in total). The solution was transferred to a 15 mL Falcon tube, and lyophilized to give a crystalline powder (65.6 mg).
[0555]The pure product was analyzed by UPLC-MS (Analytical method G; measured: 68677; expected: 68678).
example 3
Albumin-MPA-NHCH2CH2CH2—CH2CH2OCH2CH2O—CH2CH2CH2NH—C(O)CH2CH2C═O-A-R—P—R-L-S—H—K-G-P-Nle-P—F—OH
[0556]Example 3 was synthesized as example 1. The pure product was analyzed by UPLC-MS (Analytical method G, measured: 68367; expected: 68366)
[0557]The DNA fragments below were generated by standard PCR techniques using the vector pPL1146 as a template with the following primers: A 5′ primer was designed that contains a NheI site followed by sequence corresponding to the 5′ end of the human Fc contained in vector pPL1146. 3′ primers were designed to contain a EcoRI site, Apelin sequence for the appropriate construct, a glycine serine linker and sequence complimentary to the 3′ end of the human Fc contained in pPL1146. Following amplification, each of the four fragments was restriction digested with both NheI and EcoRI restriction enzymes, isolated and purified, and ligated into vector pPL1146 digested and purified in the same manner. The ligations were transform...
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